Bacillus thuringiensis isolates active against hymenopteran pests

ABSTRACT

The subject invention concerns novel microbes and genes encoding novel toxin proteins with activity against insect pests of the order Hymenoptera. Pests in the order Hymenoptera are common house pests, and they create problems in hospitals, the food industry and in agriculture. The novel Bacillus thuringiensis microbes of the invention are referred to as B.t. PS140E2 and B.t. PS86Q3. The spores or crystals of these microbes, or mutants thereof, are useful to control hymenopteran pests in various environments. The genes of the invention can be used to transform various hosts wherein the novel toxic proteins can be expressed.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (B.t.) produces an insect toxin designated asδ-endotoxin. It is synthesized by the B.t. sporulating cell. The toxin,upon being ingested in its crystalline form by susceptible insects, istransformed into biologically active moieties by the insect gut juiceproteases. The primary target is insect cells of the gut epithelium,which are rapidly destroyed.

The reported activity spectrum of B.t. covers insect species within theorder Lepidoptera, many of which are major pests in agriculture andforestry. The activity spectrum also includes the insect order Diptera,which includes mosquitos and black flies. See Couch, T. L. (1980)"Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,"Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978)"Use of Entomogenous Bacteria in Agroecosystems," Developments inIndustrial Microbiology 20:97-104. Krieg, et al., Z. ang. Ent. (1983)96:500-508, describe a B.t. isolate named Bacillus thuringiensis var.tenebrionis, which is reportedly active against two beetles in the orderColeoptera. These are the Colorado potato beetle, Leptinotarsadecemlineata, and Agelastica alni. In European Patent Application No. 0202 739 there is disclosed a novel B.t. isolate active againstColeoptera. It is known as B. thuringiensis var. san diego (B.t.sd.).U.S. Pat. No. 4,966,765 discloses the coleopteran-active Bacillusthuringiensis isolate B.t. PS86B1.

Ants comprise a large group of insects (Family Formicidae) from thetaxonomic order, Hymenoptera. They are among the most common housepests. In many situations, ants are a nuisance pest. Foraging antscreate problems with hygiene in hospitals and the food industry. Antsalso create problems in agriculture. Damage can be caused by directfeeding on plants. Harvester and fire ants are commonly associated withthis type of damage (Holldobler, B. and Wilson, E. O. 1990. The Ants,Belkap Press, Cambridge, Mass. 732p.) Some ants cause indirect damage bynurturing and protecting sap feeding insects such as mealybugs andaphids. Ants, particularly in the genus Solenopsis are capable ofproducing extremely painful stings to humans. It has been estimated thatapproximately 10,000 stings occur each year (Habermehl, G. G. 1981,Venomous Animals and Their Toxins, Springer-Verlag, N.Y., 195 p.). ThePharoah ant (Monomorium pharoanis) is primarily an urban pest. However,this species can also be an agricultural pest and damage to corn hasbeen noted (Ebeling, W. 1978. Urban Entomology, UC Press, Berkely,Calif., 695p.).

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns novel Bacillus thuringiensis (B.t.)isolates and genes therefrom which encode novel hymenopteran-activeproteins. The novel B.t. isolates, known herein as Bacillusthuringiensis PS140E2 (B.t. PS140E2), and Bacillus thuringiensis PS86Q3(B.t. PS86Q3) have been shown to be active against the Pharoah ant(Monomorium pharaonis).

The subject invention also includes mutants of the above isolates whichhave substantially the same pesticidal properties as the parent isolate.Procedures for making mutants are well known in the microbiological art.Ultraviolet light and nitrosoguanidine are used extensively toward thisend.

Further, the invention also includes the treatment of substantiallyintact cells of the novel isolates, and recombinant cells containing thegenes from the isolates, to prolong the pesticidal activity when thesubstantially intact cells are applied to the environment of a targetpest. Such treatment can be by chemical or physical means, or acombination of chemical or physical means, so long as the technique doesnot deleteriously affect the properties of the pesticide, nor diminishthe cellular capability in protecting the pesticide. The treated cellacts as a protective coating for the pesticidal toxin. The toxin becomesavailable to act as such upon ingestion by a target insect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--Photograph of a Standard SDS Polyacrylamide Gel of B.t. PS140E2,and B.t. PS86Q3.

DETAILED DISCLOSURE OF THE INVENTION

The novel Bacillus thuringiensis isolates of the subject invention havethe following characteristics in their biologically pure form:

Characteristics of B.t. PS140E2

Colony morphology--Large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Culture methods--typical for B.t.

Inclusions--Ellipse and 2 other small inclusions

Approximate molecular weight of alkali-soluble proteins (kDa)--78, 70,35.

Characteristics of B.t. PS86Q3

Colony morphology--large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Culture methods--typical for B.t.

Inclusions--1 long and 1 or 2 small inclusions

Approximate molecular weight of alkali-soluble proteins (kDa)--155, 135,98, 62, 58

A comparison of the characteristics of B. thuringiensis PS140E2 (B.t.PS140E2), and B. thuringiensis PS86Q3 (B.t. PS86Q3), and B.thuringiensis var. kurstaki (HD-1) is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Comparison of B.t. PS140E2, B.t. PS86Q3, B.t.sd., and B.t. HD-1                      B.t. PS140E2                                                                            B.t. PS86Q3  B.t. HD-1                                       ______________________________________                                        Inclusions:                                                                            Ellipse and 1 long and 1 Bipyramid                                            2 small     or 2 small                                                        inclusions  inclusions                                               Approximate                                                                            78,000      155,000      130,000                                     molecular wt.                                                                          70,000      135,000       68,000                                     of proteins                                                                            35,000       98,000                                                  by SDS-               62,000                                                  PAGE                  58,000                                                  Host range                                                                             Hymenopteran                                                                              Hymenopteran Lepidopteran                                                     and Coleopteran                                          ______________________________________                                    

The cultures disclosed in this application have been deposited in theAgricultural Research Service Patent Culture Collection (NRRL), NorthernRegional Research Center, 1815 North University Street, Peoria, Ill.61604, USA.

    ______________________________________                                        Culture       Repository No.                                                                            Deposit date                                        ______________________________________                                        Bacillus thuringiensis                                                                      NRRL B-18812                                                                              April 23, 1991                                      PS140E2                                                                       Bacillus thuringiensis                                                                      NRRL B-18765                                                                              February 6, 1991                                    PS86Q3                                                                        ______________________________________                                    

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C.122. The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of a deposit, and in any case, for a period of at least thirty(30) years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposits. All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

The B.t. isolates of the invention can be cultured using standard artmedia and fermentation techniques. Upon completion of the fermentationcycle, the bacteria can be harvested by first separating the B.t. sporesand crystals from the fermentation broth by means well known in the art.The recovered B.t. spores and crystals can be formulated into a wettablepowder, liquid concentrate, granules, or other formulations by theaddition of surfactants, dispersants, inert carriers and othercomponents to facilitate handling and application for particular targetpests. These formulation and application procedures are all well knownin the art.

Formulated products can be sprayed or applied as baits to controlhymenopteran pests.

The B.t. cells of the invention can be treated prior to formulation toprolong the pesticidal activity when the cells are applied to theenvironment of a target pest. Such treatment can be by chemical orphysical means, or by a combination of chemical and/or physical means,so long as the technique does not deleteriously affect the properties ofthe pesticide, nor diminish the cellular capability in protecting thepesticide. Examples of chemical reagents are halogenating agents,particularly halogens of atomic no. 17-80. More particularly, iodine canbe used under mild conditions and for sufficient time to achieve thedesired results. Other suitable techniques include treatment withaldehydes, such as formaldehyde and glutaraldehyde; anti-infectives,such as zephiran chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Bouin's fixative and Helly'sfixative (See: Humason, Gretchen. L., Animal Tissue Techniques, W. H.Freeman and Company, 1967); or a combination of physical (heat) andchemical agents that prolong the activity of the toxin produced in thecell when the cell is applied to the environment of the target pest(s).Examples of physical means are short wavelength radiation such asgamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like.

The toxin genes of the subject invention can be introduced into a widevariety of microbial hosts. Expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the situs of hymenopteran insects where they willproliferate and be ingested by the insects. The result is a control ofthe unwanted insects. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinproduced in the cell. The treated cell then can be applied to theenvironment of target pest(s). The resulting product retains thetoxicity of the B.t. toxin.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the "phytosphere"(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

A wide variety of ways are available for introducing the B.t. geneexpressing the toxin into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. One can providefor DNA constructs which include the transcriptional and translationalregulatory signals for expression of the toxin gene, the toxin geneunder their regulatory control and a DNA sequence homologous with asequence in the host organism, whereby integration will occur, and/or areplication system which is functional in the host, whereby integrationor stable maintenance will occur.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In some instances, it may bedesirable to provide for regulative expression of the toxin, whereexpression of the toxin will only occur after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger, particularly by using an active promoter, as well as byemploying sequences, which enhance the stability of the messenger RNA.The initiation and translational termination region will involve stopcodon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5' or 3' of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon(s), thepolyadenylation signal sequence, if any, and the terminator region. Thissequence as a double strand may be used by itself for transformation ofa microorganism host, but will usually be included with a DNA sequenceinvolving a marker, where the second DNA sequence may be joined to thetoxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototropy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the toxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the toxin-producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, and usually not more than about 1000 bp of asequence homologous with a sequence in the host. In this way, theprobability of legitimate recombination is enhanced, so that the genewill be integrated into the host and stably maintained by the host.Desirably, the toxin gene will be in close proximity to the geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that a toxin gene islost, the resulting organism will be likely to also lose thecomplementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the lambda left and right promoters, the tac promoter,the naturally-occurring promoters associated with the toxin gene, wherefunctional in the host. See for example, U.S. Pat. Nos. 4,332,898,4,342,832 and 4,356,270. The termination region may be the terminationregion normally associated with the transcriptional initiation region ora different transcriptional initiation region, so long as the tworegions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pRO1614, and the like. See for example, Olson et al., (1982) J.Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, andU.S. Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.

The B.t. gene can be introduced between the transcriptional andtranslational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants then can be tested forpesticidal activity.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility of toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion; ease of killing and fixingwithout damage to the toxin; and the like. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,Escherichia coli, Bacillus subtilis, Streptomyces lividans, and thelike.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the recombinant microbial cell can be done as disclosedinfra. The treated cells generally will have enhanced structuralstability which will enhance resistance to environmental conditions.Where the pesticide is in a proform, the method of inactivation shouldbe selected so as not to inhibit processing of the proform to the matureform of the pesticide by the target pest pathogen. For example,formaldehyde will crosslink proteins and could inhibit processing of theproform of a polypeptide pesticide. The method of inactivation orkilling retains at least a substantial portion of the bio-availabilityor bioactivity of the toxin.

The cellular host containing the B.t. insecticidal gene may be grown inany convenient nutrient medium, where the DNA construct provides aselective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells may be formulated in a variety of ways. They may beemployed as wettable powders, baits, granules or dusts, by mixing withvarious inert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the natureof the particular formulation, particularly whether it is a concentrateor to be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the hymenopteranpest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling,baits or the like.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Culturing B.t. Isolates

A subculture of the B.t. isolate can be used to inoculate the followingmedium, a peptone, glucose, salts medium.

    ______________________________________                                        Bacto Peptone          7.5    g/l                                             Glucose                1.0    g/l                                             KH.sub.2 PO.sub.4      3.4    g/l                                             K.sub.2 HPO.sub.4      4.35   g/l                                             Salt Solution          5.0    ml/l                                            CaCl.sub.2 Solution    5.0    ml/l                                            Salts Solution (100 ml)                                                       MgSO.sub.4.7H.sub.2 O  2.46   g                                               MnSO.sub.4.H.sub.2 O   0.04   g                                               ZnSO.sub.4.7H.sub.2 O  0.28   g                                               FeSO.sub.4.7H.sub.2 O  0.40   g                                               CaCl.sub.2 Solution (100 ml)                                                  CaCl.sub.2.2H.sub.2 O  3.66   g                                               pH 7.2                                                                        ______________________________________                                    

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and crystals, obtained in the above fermentation, can beisolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Testing of B.t. Isolates

Broths were tested for the presence of β-exotoxin by a larval house flybioassay (Campbell, D. P., Dieball, D. E. and Bracket, J. M., 1987,Rapid HPLC assay for the β-exotoxin of Bacillus thuringiensis. J. Agric.Food Chem. 35:156-158). Only isolates which tested free of β-exotoxinwere used in the assays against ants.

A bait was made consisting of 10% Bacillus thuringiensis isolates of theinvention and Crosse and Blackwell mint apple jelly. Approximately 100ants were placed in each plastic test chamber replicate with the baits.Control experiments were performed with untreated mint apple jelly. Eachtest was replicated a minimum of 10 times. Mortality was assessed at 7,14 and 21 days after introduction of the bait to the ants. Results areshown below:

    ______________________________________                                        Toxicity of B. thuringiensis Isolates to the Pharoah Ant                      (Monomorium pharoanis)                                                        B.t. Isolate                                                                              Percent Mortality (S.D.)*                                         ______________________________________                                        PS140E2     91 (9)                                                            PS 86Q3      84 (20)                                                          Control     11 (4)                                                            ______________________________________                                         *S.D. = Standard Deviation                                               

EXAMPLE 3 Insertion of Toxin Gene Into Plants

The genes coding for the insecticidal toxin, as disclosed herein, can beinserted into plant cells using the Ti plasmid from Agrobactertumefaciens. Plant cells can then be caused to regenerate into plants(Zambryski, P., Joos, H., Gentello, C., Leemans, J., Van Montague, M.and Schell, J [1983] Cell 32:1033-1043). A particularly useful vector inthis regard is pEND4K (Klee, H. J., Yanofsky, M. F. and Nester, E. W.[1985] Bio/Technology 3:637-642). This plasmid can replicate both inplant cells and in bacteria and has multiple cloning sites for passengergenes. The toxin gene, for example, can be inserted into the BamHI siteof pEND4K, propagated in E. coli, and transformed into appropriate plantcells.

EXAMPLE 4 Cloning of Novel B. thuringiensis Genes Into Baculoviruses

The genes of the invention can be cloned into baculoviruses such asAutographa californica nuclear polyhedrosis virus (AcNPV). Plasmids canbe constructed that contain the AcNPV genome cloned into a commercialcloning vector such as pUC8. The AcNPV genome is modified so that thecoding region of the polyhedrin gene is removed and a unique cloningsite for a passenger gene is placed directly behind the polyhedrinpromoter. Examples of such vectors are pGP-B6874, described by Pennocket al. (Pennock, G. D., Shoemaker, C. and Miller, L. K. [1984] Mol.Cell. Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, G.E., Summers, M. D. and Fraser, M. J. [1983] Mol Cell. Biol.3:2156-2165). The genes coding for the protein toxins of the inventioncan be modified with BamHI linkers at appropriate regions both upstreamand downstream from the coding region and inserted into the passengersite of one of the AcNPV vectors.

We claim:
 1. A process for controlling hymenopteran insect pests whichcomprises contacting said insect pests with an insect-controllingeffective amount of Bacillus thuringiensis PS140E2 having theidentifying characteristics of NRRL B-18812, including activity againsthymenopteran pests, or Bacillus thuringiensis PS86Q3 having theidentifying characteristics of NRRL B-18765, including activity againsthymenopteran pest.
 2. The process, according to claim 1, wherein saidhymenopteran pest is the Pharaoh ant.
 3. A process for controllinginsects pests of the order Hymenoptera which comprises(1) preparing abait comprising Bacillus thuringiensis PS140E2, or Bacillusthuringiensis PS86Q3, or spores or crystals thereof; and (2) placingsaid bait in areas visited by hymenopteran pests.
 4. A process,according to claims 1 or 3, wherein the Bacillus thuringiensis cells aretreated, while substantially intact, to prolong the pesticidal activitywhen the substantially intact cells are applied to the environment ofsaid insect pests.
 5. A composition of matter comprising Bacillusthuringiensis PS140E2, or Bacillus thuringiensis PS86Q3, or spores orcrystals thereof, in association with an insecticide carrier.
 6. Abiologically pure culture of Bacillus thuringiensis PS140E2, having theidentifying characteristics of NRRL B-18812, including activity againsthymenopteran pests, or Bacillus thuringiensis PS86Q3, having theidentifying characteristics of NRRL B-18765, including activity againsthymenopteran pests.